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Expanding the Roles of tRNA Thiolation in Bacteria: Involvement of 2‐thiouridine tRNA in Signaling Bacterial Sulfur Metabolism
Author(s) -
Black Katherine Ann,
Kus Nicole Joanne,
Dos Santos Patricia Coutinho
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.807.3
Subject(s) - biochemistry , transfer rna , amino acid , methionine , cysteine , sulfur metabolism , metabolism , biology , protein biosynthesis , amino acid synthesis , biosynthesis , translation (biology) , chemistry , operon , gene , escherichia coli , enzyme , lysine , rna , messenger rna
Post‐transcriptional modifications of tRNA are found in all organisms and are critical for their structure and function during translation. 2‐thiouridine (s 2 U) modification at the wobble position of tRNA Glu, Gln, Lys is required for accurate and efficient protein synthesis and its absence impacts cellular viability. In addition to its role in translation, in yeast, the s 2 U modification is used as a sensor to gauge nutrient availability and regulate translational capacity and amino acid homeostasis, in which s 2 U abundance reflects the availability of sulfur‐containing amino acids, cysteine and methionine. In previous work, we showed that B. subtilis s 2 U biosynthesis involves only two proteins: the cysteine desulfurase YrvO and the thiouridylase MnmA. Genes coding for both proteins are located in an operon directly downstream of the master regulator of cysteine metabolism, cymR , suggesting a link between s 2 U formation and sulfur amino acid metabolism in this bacterium. In this study we investigated s 2 U's role in metabolic homeostasis and translational regulation as a sensor of sulfur availability in bacteria. B. subtilis wild‐type was cultured in minimal media containing varying concentrations of an individual sulfur source: sulfate, cysteine, or methionine. Using our established LC‐MS method, analysis of tRNA‐thionucleosides showed that the abundance of s 2 U correlates to the concentration, but not the source, of sulfur available in the growth media. This relationship appears to be specific to s 2 U, as the levels of an additional thionucleoside, s 4 U, remained constant across all conditions; supporting the notion that only s 2 U is responsive to sulfur availability. Western blot analysis of YrvO and MnmA in these cultures showed patterns consistent with those of CymR expression. That is, higher expression of YrvO and MnmA was observed in cultures using sulfate, cysteine or cystine as the sole sulfur source when compared to thiosulfate, methionine or homocystine. Surprisingly, expression levels of YrvO and MnmA did not correlate with the concentration of sulfur source, or accumulation of s 2 U thionucleoside, suggesting that the activity of these enzymes is likely controlled by the concentration of cysteine, which affects the catalytic turnover rate of YrvO, the first enzyme in the s 2 U pathway. In this model, the availability of sulfur controls the flux of sulfur transfer which impacts the overall levels of the signaling metabolite s 2 U tRNA. Current work is aimed towards evaluating whether s 2 U levels dictate expression of proteins involved in translation and growth specific processes. Furthermore, these analyses are being expanded to other bacterial species to potentially establish this regulatory role across a diverse group of bacteria. Support or Funding Information This work has been funded through the Wake Forest University Center for Molecular Communication and Signaling graduate fellowship.